Matrices comprising a modified polysaccharide

ABSTRACT

The present invention discloses a matrix comprising a modified polysaccharide consisting of repeating disaccharide units whereby in at least 11% of the disaccharide units one primary alcohol group is oxidized into a carboxylic acid group.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 2, 2016, isnamed 4348-107US_SL.txt and is 998 bytes in size.

FIELD OF THE INVENTION

The present invention relates to matrices comprising a modifiedpolysaccharide and the applications of such matrices. The matrixes canbe used as scaffold for living cells regenerative implants, plasticsurgery implants and controlled drug release implants. In additionthereto, the matrixes of the present invention are suitable for use as afood additive, a component for cosmetic compositions and for otherindustrial purposes.

BACKGROUND OF THE PRESENT INVENTION

In the text of the present application, the nomenclature of amino acidsand of peptides is used according to “Nomenclature and symbolism foramino acids and peptides”, Pure & Appl. Chem., Vol. 56, No. 5, pp.595-624, 1984, if not otherwise stated.

The following abbreviations have the meaning as given in the followinglist, if not otherwise stated:

AFM atomic force microscopyCD circular dichroismCSF cell shape factorCT computed tomographyDIC differential interference contrast imagingDLS dynamic light scatteringDMEM Dulbecco's modified Eagle's mediumDNA deoxyribonucleic acidECM extracellular matrixEDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlorideESEM environmental scanning electron microscopyFTIR Fourier transform infrared spectroscopyHCl hydrochloric acidKBr potassium bromideMAS-NMR magic angle spinning NMRMD molecular dynamicsMES 2-(N-morpholino)ethanesulfonic acidMn number average molecular weightMRI magnetic resonance imagingMWCO molecular weight cut offNaOCl sodium hypochlorideNaOH sodium hydroxideNMR nuclear magnetic resonancePCR polymerase chain reactionPDB protein databaseRNA ribonucleic acidRMSD root mean square deviationSEM scanning electron microscopysiRNA small interfering RNASLS static light scatteringTEMPO (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl.

Living cells of higher organisms reside in an environment that ismechanically and biologically well-defined by an extracellular matrix(in the following ECM). Structural and mechanical aspects of the ECMsuch as stiffness and topography can have a substantial influence ondifferent cell functions like cell growth or differentiation of thecells.

The present invention provides matrices wherein important mechanical andchemical properties of the matrix can be adjusted according to thedesired application. Thus, it can be for example required to lower theshear modulus G′ of the matrix or to improve the optical propertiestowards an increased transparency of the matrix.

The cell surrounding has been considered in the past decades to be animportant piece of the puzzle of organogenesis. It is known that eachtype of cell builds and evolves in a specific environment that providesthe mechanical properties and the nutritional needs of the cell. Thisenvironment is called extracellular matrix (ECM). Structural andfunctional components of the ECM can modulate cell behavior and functionand also determine which cell can interact with another in the humanbody. Nevertheless, most of these intercellular interactions are notfully understood. It has been shown that the properties of the ECMsupporting the cell in the human body have many aspects (physical andchemical) that have been shown to impact the cell fate in vitro and invivo. It has been found that in addition to cell adhesion moieties andgrowth factors, the physical attributes of a cellular microenvironmentnamely, stiffness and topography are another important element indictating and controlling cell fate and function.

Providing a synthetic ECM would be a significant contribution toinvestigation of such intercellular interaction. Importantly, a modelemploying such ECM would allow mimicking of human tissues in vitro andbe amenable to translation in vivo. A further aim of this model is toenable communicating with the body and aid in healing or regeneration oftissues. Injectable, biologically well-defined matrixes with tailor-madeand tunable physical properties would be the evolutionary next-step insynthetic niches for cells.

The present invention provides a matrix suitable for these applications.Moreover, the matrix of the present invention can also be used as a foodadditive, material for surgery implants and controlled drug releaseimplants, as lubricant for industrial purposes as well as forconditioning of liquids.

In the present application the matrix is defined as molecules composingthe cell surrounding. In general matrix is made up of differentcomponents that can be classified as:

(1) the soluble molecules, e.g. growth factor and other signalingmolecules and(2) the structural polymers, composed of proteins and polysaccharidesthat determines the mechanical properties of the tissues.

The term “growth factor” relates to a naturally occurring compound whichis capable of stimulating cellular growth, proliferation anddifferentiation. Preferably, the growth fact is a polypeptide or aprotein, for instance, a water-soluble protein.

The term “protein” relates to a polymeric structure which consists ofone or several polypeptides. Polypeptides, in turn, consist of aminoacid residues joined together by peptide bonds. Preferably, the aminoacids of the proteins are La-amino acids, whereby proteinogenic aminoacids are particularly preferred. It is preferred that proteins actingas components of an extracellular matrix are not water-soluble. The term“peptide sequence” used in the present invention relates to apolypeptide. Preferably, a peptide sequence has between 2 and 50 aminoacid residues, particularly preferred between 5 and 20 amino acidresidues.

In the present application the term “polysaccharide” relates to apolymeric carbohydrate structure which is formed of repeating unitsjoined together by glycosidic bonds. Preferably, the repeating units areeither mono- or disaccharides and the polymeric structure of thepolysaccharide is non-branched. It is preferred that the number averagemolecular weight of the polysaccharide ranges from 10 000 Da to 500 000Da, particularly preferred the number average molecular weight of thepolysaccharide ranges from 50 000 Da to 300 000 Da, whereby the numberaverage molecular weight of the polysaccharide ranging from 80 000 Da to140 000 Da is even more preferred.

Some synthetic matrixes for use as ECM have already been commercialized.So far three different strategies have been explored to producesynthetic ECMs:

(1) Use of animal protein extracts such as Matrigel® which suffer from alack of batch to batch reproducibility and a poor definition of thecomponents. This leads to difficulties in the interpretation of theresults obtained with such ECMs.

(2) Use of synthetic polymers such as degradable polyester that althoughbiocompatible are not easy to synthesize and manufacture andadditionally, require knowledge in synthetic chemistry to set it up andare also difficult to translate into in vivo clinical settings.

(3) Use of natural components of the ECM such as collagen or hyaluronicacid, which reproduce only one aspect of the natural ECM environment.

As outlined in the review of Tan et a! (Materials 2010, 3, 1746-1767)various polysaccharides have been suggested as suitable materials forECMs in the last decades. Prominent among them are hyaluronic acid,alginate acid and chitosan. Hyaluronic acid when modified using longchain alcohols can yield gels that are formed due to physicalcross-links established by the aggregations of the hydrophobic alkylchains in water. Hyaluronic acid can also be gelled using covalentcrosslinking. In this case, the hyaluronic acid is oxidized to bearaldehyde groups which are then reacted with N-succinyl modified chitosanor other biopolymers. Subsequently, the crosslinking is induced bydiamine through Schiff base formation. Alginic acid can also beprocessed into gels that can serve as cell supports by ioniccrosslinking it with divalent cations Ca²⁺, Mg²⁺, Ba²⁺ or Sr²⁺.

SUMMARY OF THE INVENTION

The present invention relates to a matrix comprising a modifiedpolysaccharide consisting of repeating disaccharide units whereby in atleast 11% of the disaccharide units one primary alcohol group isoxidized into a carboxylic acid.

It is an essential component of the matrix of the present invention tocontain at least one modified polysaccharide whereby the modifiedpolysaccharide consists of repeating disaccharide units. In a preferredembodiment the modified polysaccharide is derived from agarose. In yetanother preferred embodiment the modified polysaccharide is derived fromfragmented agarose.

Agar, the main source of agarose is a structural polysaccharide of thecell walls of a variety of red algae. Important sources of agar areGelidiaceae such as Gelidium amansii, Gelidium japonicum, Gelidiumpacificum, Gelidium subcostatum, Pterocladia tenuis and Acanthopeltisjaponica, red algae belonging to Gracilariaceae such as Gracilariaverrucosa and Gracilaria gigas, red algae belonging to Ceramiaceae suchas Ceramium kondoi and Campylaephora hypnaeoides. Agar consists of twogroups of polysaccharides, namely agarose and agaropectin. Agarose is aneutral, linear polysaccharide with no branching and has a backboneconsisting of 1,3-linked β-D-galactose-(1-4)-α-L-3,6 anhydrogalactoserepeating units. This dimeric repeating unit, called agarobiose differsfrom a similar dimeric repeating unit called carrabiose which is derivedfrom carrageenan in that it contains 3,6-anhydrogalactose in the L-formand does not contain sulfate groups.

Such dimeric repeating units derived from naturally occurringpolysaccharides are chemically modified by the regioselective oxidationof the primary alcohol group to a carboxylic acid group. Such oxidationcan conveniently carried out by sodium hypochloride in the presence of2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO). The reaction mechanism isshown in Scheme 1 below. It goes without saying that the regioselectiveoxidation of the primary alcohol group to the carboxylic acid group canbe also performed by other reactions which are well-known to a personskilled in the art as long as these reactions are sufficiently selectiveand no undesired oxidation of the secondary alcohol group and of otherfunctionalities of the polysaccharides takes place. In addition, theoxidation of the primary alcohol groups of the polysaccharide can alsobe carried out by an enzymatic process or upon using a bacteriologicalsystem.

Depending on the chosen reaction conditions it is possible to oxidizeonly a certain percentage of the primary alcohol groups of thepolysaccharide. According to the invention at least 11% of thedisaccharide repeat units are oxidized. In a preferred embodiment atleast 20 up to 99% of the disaccharide repeat units of the primaryalcohol groups are oxidized into the carboxylic acid group. In aparticularly preferred embodiment in 50-95% of the disaccharide repeatunits the primary alcohol group is oxidized into the carboxylic acidgroup. In yet another embodiment more than 75% of the disacchariderepeat units the primary alcohol group is oxidized to the carboxylicacid group.

Thus, the oxidation reaction is carried out in a controlled manner sothat only a partial oxidation of the primary alcohol groups takes place.However, the polysaccharide can also be oxidized in such a way thatabout 100% of the primary alcohol groups are oxidized.

The completely or partially oxidized polysaccharide can be subsequentlyblended with an unmodified polysaccharide which may either be the samepolysaccharide or another polysaccharide. Since the nature and extend ofthe chemical modification of the modified polysaccharide can beprecisely controlled and the blending ratio with another polysaccharideor the same unmodified polysaccharide can be adjusted it is possible tocontrol the chemical properties of the resulting matrix.

The weight ratio of the modified polysaccharide in the matrix of thepresent invention is in the range of 1-99 wt.-%. In a preferredembodiment the present invention the weight ratio of the modifiedpolysaccharide in the matrix is greater than 1 wt.-%, preferably it isgreater than 10 wt.-%. It is yet even more preferred that the ratio ofthe modified polysaccharide in the matrix is greater than 20 wt. %, inparticular greater than 50 wt.-%.

One important aspect of the present invention is the shear modulus G′ ofthe matrix. According to the present invention the shear modulus canrange from about 10 Pa which reflects the structure of a nerve tissue toabout 10⁷ Pa which corresponds with the shear modulus of cartilagetissues. By blending gels of different extent of chemical modificationthe nanoscale structure of the gel can be impacted. It has been shownthat nanoscale topography influences cell shape, cytosceletal assemblyand function. It is for this reason that the matrix of the presentinvention can induce changes in mammalian cell shape or mammalian cellfunction.

The shear modulus of the matrix ranges preferably from 1 Pa to 100 kPa,more preferred from 1 Pa to 50 kPa and in a most preferred embodiment ina range from 10 Pa to 10 kPa, whereby the measurement of the shearmodulus is carried out at a temperature of 37° C. as specified below.

In an especially preferred embodiment agarose wherein the primaryalcohol group has been oxidized in a carboxylic acid group is blendedwith non-modified agarose.

Agarose is commonly used for separation techniques such aselectrophoresis, Gel Permeation Chromatography, High Performance Liquidchromatography but also as a food additive. Recently the use of agarosehydrogel has shown to be useful for engineered ECM. Agarose has beensuccessfully used to engineer cartilage de novo which suggest thatfurther development will offer the possibility to regenerate othertissues. It has been shown that agarose gels induce bone reconstructionin vivo. It has also been shown that it is possible to modify theagarose backbone by oxidizing the primary alcohol group of theD-galactose residue in a highly regioselective manner. This modificationenables grafting of molecules on the agarose gel through a carboxylicacid group. Therefore, the matrix of the present invention is highlyvaluable for biochemical and medical applications.

It has been observed that the oxidation of the C6 primary alcohol groupof the D-galactose residue leads to a decrease of shear modulus but alsoto a weaker gel. The design of new material using agarose has beeninvestigated by creating copolymer of agarose-collagen, oragarose-cellulose, but also by blending agarose with natural polymer ofthe ECM.

Modified agarose is the preferred component of the matrix of the presentinvention. It is however equally preferred that unmodified agarose isused as a matrix component. Moreover, it is possible to use otherpolysaccharides of natural origin as component which can be blended withmodified agarose if such component cannot be oxidized as describedabove. If the structure of the polysaccharide contains a primary alcoholgroup which can be oxidized to a carboxylic acid group the repeatingdisaccharide units can be modified as described above in more detail foragarose. Other polysaccharides which can be used in the presentinvention are listed in Table 1 below. Polysaccharides in cells withbold borders comprise disaccharide units having a primary alcohol group.Therefore, these polysaccharides can be chemically modified in themanner described above. In particular, the polysaccharides used for thematrix of the present invention are hyaluronic acid, heparin sulfate,dermatan sulfate, chondroite sulfate, alginate, chitosan, pullulan,k-carrageenan. In the most preferred embodiment agarose is used.

In another embodiment of the present invention the matrix containscarrageenans in modified or/and unmodified form. Carrageenans arepolysaccharides that are contained in red algae belonging toGigartinaceae, Solieriaceae, Hypneaceae and the like. K-carrageenan,A-carrageenan and q-carrageenan are known.

TABLE 1 Gelation Name Structure Origin mechanism Hyaluronic Acid

Mammalian use of crosslinker to form a 3D network Heparin sulfate

Mammalian Dermatan sulfate

Mammalian Chondroite sulfate

Mammalian Alginate

Algae Ca²⁺ bridges Chitosan

Marine shell Repulsion of charges Pullulan

Fungus Like cellulose, sheet organization k-Carrageenan

Algae helices aggregation Agarose

Algae helices aggregation /

In further preferred embodiments of the present invention the carboxylicgroup which is derived from the oxidation of the primary alcohol groupis covalently coupled with a peptide sequence. In preferred embodimentsthe peptide sequence is selected from the group consisting of the celladhesion sequence arginine-glycine-aspartic acid, the peptide sequencesIKVAV (SEQ ID NO: 1) and YIGSR (SEQ ID NO: 2) or a protein which ispreferably selected from collagen, collagen fragments, fibronectin andmixtures thereof. In yet another embodiment the protein sequence isvitronectin.

In another preferred embodiment the matrix may contain a modification ofthe carboxylic acid group derived from the oxidation of the primaryalcohol group insofar that this carboxylic acid group is covalentlylinked to a nucleic acid sequence. The nucleic acid sequence may besingle-stranded DNA, double-stranded DNA, single-stranded RNA and siRNA.The linkage between the carboxylic acid group and nucleic acid can beintroduced by a method of click chemistry as known in the prior art. Incase single-stranded nucleic acids are linked to the matrix, suchsingle-stranded molecules may hybridize to complementary single-strandedmolecules which are linked to other components. This offers theopportunity to easily attach molecules or even whole cells to thematrix.

Depending on the purpose of the use of the matrix it may be particularlyhelpful to include specific points of fixation into the matrix which canbe designed according to the intended use of the matrix.

The present disclosure further describes the ability to change thetertiary structure of polysaccharides in a manner that the roughness,stiffness, thermal gelation behavior and optical feature of the materialcan be finely tuned to target a specific material, whereby themodification made on the backbone consists of an oxidation of theprimary alcohol group of agarose backbone to the carboxylic acid group.It was demonstrated that this modification induces a change oforganization of the polymer backbone and has been replicated ink-carrageenan as another polysaccharide. The gel of interest can beobtained by controlling the amount of chemical modification of thebackbone but also by blending the native polysaccharide with themodified polysaccharide. This results in a uniform material with featuresuch as stiffness, thermal gelation and roughness that can be adjustedby incorporating different amounts of each polysaccharide.

The ease of control of the physical properties enables the design of anenvironment which is mechanically similar to natural human tissue butalso biologically neutral since the agarose does not interact withcells. In case it is desired that cells interact with the material,biological ligands that are recognized by cells have to be grafted onthe polymer backbone. Therefore two strategies have been followed; firstthe chemical way by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDC) coupling that allows direct chemical binding of thecarboxylic acid group of the polysaccharide to the amine group on theN-terminus of a peptide. This process is illustrated in Scheme 2.

The peptide sequence can also be attached to the modified polysaccharideby click chemistry, whereby either azide or the alkyne moiety is coupledto the modified polysaccharide and the other moiety is coupled to thepeptide sequence.

The second approach has been made with DNA coupling that enables aneasier manipulation for the final user. An oligo-DNA strand can becoupled to the polymer backbone with the complementary oligo-DNA strandthat has been previously chemically bound to the peptide of interest.This way of binding is for the final user a step forward to a fullytunable system, where the mechanical properties can be adjusted bymixing two components (native with modified polysaccharide) and thebiological signal incorporated to the backbone polymer by adding acomponent to the system (the complementary oligo-DNA strand bounded tothe peptide of interest). This principle is shown in FIG. 3.

In a preferred embodiment the matrix can be used as a scaffold of livingcells in order to grow cells in a three-dimensional structure whichresembles the natural enviroment. Cells, preferably human cells, whichare preferably selected from the group consisting of chondrocytes,osteoblasts, osteoclast, skin epithelial cells, intestinal epithelialcells, corneal epithelial cells, astrocytes, neurons, oligodendrocytes,smooth muscle cells, endothelial cells, cardiomyocytes, pancreatic isletcells, kidney epithelial cells, embryonic stem cells, pluripotent stemcells; or naïve cells obtained from umbilical cord.

The matrix of the present invention can be further used for experimentalpurposes since the interaction of cells and its understanding gains moreand more importance in many fields of biological research.Alternatively, the matrix can be used in order to produce artificialtissues. It is for example possible to grow the cells as described abovein order to produce artificial three-dimensionally linked tissues whichcan be used as implant for the curing of various defects. It is forexample possible to produce homologous bone structures by cultivatingosteoblasts and/or osteoclasts in the matrix as described herein.Alternatively, artificial skin or cartilage can be produced. Since thematerial is well compatible with the immune system, no unexpectedallergic reactions can be expected. This is especially true whenhomologous cells are used for the preparation of artificial tissues.

The matrix of the present invention can be implanted in mammalian bodycavities both in the presence and in the absence of growth factors.These cavities can be with or without cells.

In a particularly preferred embodiment the matrix of the presentinvention is used as a regenerative implant. Such a regenerative implantis produced in vitro by using the matrix as a scaffold for the tissueswhich grow three-dimensionally in vitro. After the implant has reachedthe desired structure it can be implanted into a patient. Since the formof such an implant can be precisely designed by using the appropriatestiffness and viscosity or the required modulus usually in the firststep the matrix is formed as a scaffold having the desired form. Thismatrix may be present in various forms. Sheets with a defined thicknessare incubated with dermal cells and artificial skin can be producedthereby. Tubes having a well-defined diameter are incubated withsuitable cells which form blood vessels like endothelial cells in orderto produce artificial artherials or veins.

It is possible to form tubular structures which are brought into contactwith such type of cells which form blood vessels. Alternatively, thematrix may have the form of a disc and the matrix will be brought intocontact with cells which form cartilage tissues. Since the mechanicalproperties like stiffness, rigidity and viscosity of the matrix can becontrolled by selecting the appropriate ratio of modifiedpolysaccharide:unmodified polysaccharide, in particular modifiedagarose:unmodified agarose, the properties can be regulated veryprecisely. It is a further advantage of the present invention that thethree-dimensional structure of the tissue is given by the matrix.Therefore, thickness, length or any other desired form of the matrix canbe prepared by using a suitable form or mold. For medical purposes it ispreferred to sterilize the agarose. This can be done either byappropriate chemicals or more preferred by heat treatment or byradiation.

The methods used to sterilize the agarose are not particularly limited.As a suitable chemical agent for sterilization ethylene oxide isadvantageously used. Agarose can also be sterilized by a treatment withionizing radiation, such as x-rays, γ-radiation or with electronic beam.It is however, particularly preferred to carry out sterilization ofagarose by a heat treatment. Such heat treatment is advantageouslycarried out in an autoclave at a temperature of ca. 121° C. and pressureof 1.1 bar whereby the sterilization time of at least 15 min isrequired. Alternatively, sterilization can be carried out by afiltration using a sterile filter with pore size of less than 0.5 μm,preferably less than 0.3 μm.

After the matrix has been brought into the desired form and the form hasbeen sterilized it is brought into contact with the desired cells,preferably in the appropriate medium which contains the desired growthfactors. Depending on the type of cell which grows on the matrix,appropriate cytokines are added. It is also possible to add sequentiallytwo different types of cells in order to form an matrix wherein tissueshave been grown which resemble the part of the body which should bereplaced or supported as far as possible. Such matrices wherein tissuecells have been grown to form tissues can be used as regenerativeimplants in the treatment of humans. It is possible to produce by usingthe matrix of the present invention regenerative implants which can beused as artificial skin, as artificial blood vessels or for thereplacement of nerve tissues. It is also possible to produce mucosaltissues or parts of the eye, in particular artificial lenses. Aparticular advantage of the present invention is that the matrix hassuperior optical quality which means that the matrix can be completelyclear. In particular, a matrix containing a modified polysaccharidehaving a high modification degree has a particularly high transparency.This is extremely important for forming artificial cornea and/or lenses.

In another embodiment of the present invention the matrix can be used asa plastic surgery implant for reconstructive and cosmetic surgery indiverse body regions: in the facial region such as a nose, a forehead, ajaw, a cheek but also in a breast, a hip, a calf and the like. Forexample, in case of the nose correction, because the small implant isinserted between the nasal bone and the periosteum, the material of theimplant need to be sufficiently stiff so that the implant does notdeviate from its original position. However, in case of the breastcorrection, a relatively large implant and soft implant is employed.

The mechanical properties of the matrix such as stiffness and roughnesscan be adjusted as outlined above. Thus, the properties of the plasticsurgery implant comprising the matrix can be conveniently adjusteddepending on the intended purpose and used in a wide variety of bodyregions.

In yet another embodiment of the present invention the matrix can beused for producing controlled drug release formulations ofpharmaceutically active components. By varying the mechanical propertiesof the matrix the controlled drug release formulation can be tailored tothe desired purpose. If for example the matrix is very stiff,pharmaceutical agents which are included within the matrix will bedelivered after implantation into the body very slowly. On the otherhand, if the viscosity and the stiffness of the matrix are rather low, apharmaceutical agent which is entrapped in the matrix will be releasedrather quickly.

In a further embodiment of the present invention the matrix can be usedwith pharmaceutically active agents like growth factors, insulin,biologically active peptides, chemokines, cytokines, steroids,antibiotics, analgethics and anti-inflammatory agents or anti-cancerdrugs.

In a further embodiment the matrix can also be used for diagnosticpurposes by including imaging agents as e.g. magnetic resonance imaging(MRI) contrast agents, computed tomography (CT) contrast agents,fluorescent imaging probes or radionuclei. By including those agentsinto the matrix and applying thereafter the matrix to the human body theagents are trapped into the matrix and can be released in a controlledmanner by adjusting the properties of the matrix like viscosity,stiffness and form which depends on the intended use.

It is also possible to include into the matrix cells which form a tissueand pharmaceutically active agents.

Another embodiment of the present invention relates to the use of thematrix as a food additive. Such food additive is useful for thepreparation of foods, drinks or seasonings. Preferably, the foods,drinks or seasonings contain 0.1 to 30 wt.-% of the matrixpolysaccharide relative to the total weight of said food, drink orseasoning, more preferably they contain 0.5 to 25 wt.-% of the matrixpolysaccharide, whereby the content of 1 to 20 wt.-% of the matrixpolysaccharide is particularly preferred. The foods, drinks orseasonings containing the matrix of the present invention are notspecifically limited. For instance, examples of such food include thefollowing: products of processed cereal (e.g., wheat flour products,starch products, premixed puddings, jam, buckwheat noodle, wheat-glutenbread, jelly bears, gelatine noodle and packed rice cake), products ofprocessed fat and oil (e.g., margarine, salad oil, mayonnaise anddressing), products of processed soybeans (e.g., tofu, miso andfermented soybean), products of processed meat (e.g. brawn and sausage),processed marine products (e.g., frozen fish, fish paste and fishfingers), dairy products (e.g., raw milk, cream, yogurt, butter, cheese,condensed milk, powdered milk and ice cream), products of processedvegetables and fruits (e.g., paste, jam, pickle), and the like.

Because the components of the matrix have a sufficient chemicalstability, the process for producing the food, drink or seasoningcontaining the matrix of the present invention is not limited to aspecific one. Any processes including cooking, processing and othergenerally employed processes for producing a food, drink or seasoningcan be used and the components of the matrix may be added before, duringor after the cooking or processing, either separately or in a form of apre-prepared matrix.

Importantly, the amount as well as the properties of the matrix employedproducing the food, drink or seasoning can be adapted according to thedesired consistency of the resulting products. For instance, preparationof jelly bears typically requires a higher content of the matrixpolysaccharides than preparation of a pudding.

The matrix of the present invention can also be used as a pharmaceuticalexcipient, for instance for the preparation of oral pharmaceuticalformulations.

In another embodiment the matrix of the present invention is used as acomponent of cosmetic compositions such as make-up, blush, lipstick,eyeshadow, antiperspirants, deodorants and concealer. The cosmeticcompositions comprise 0.1 to 50 wt.-% of matrix polysaccharide,preferably 0.5 to 30 wt.-% of matrix polysaccharide, even more preferred1 to 25 wt.-% and particularly preferred 2 to 20 wt.-%. The content ofmatrix polysaccharide is chosen according to the desired mechanicalproperties of the cosmetic compositions. Preferably, the cosmeticcompositions are characterized by being single phase.

Preferably, the cosmetic compositions are solid or semi-solid attemperature of 25° C. and have such a consistency that they can bemolded into the form of a stick. For this purpose, the compositions canbe heated until molten and then poured into a mold and cooled.Alternatively, the compositions are capable of being formed into sticks,but are poured into pans or other types of cake or cream forms todeliver certain consumer benefits. For example, an eyeshadow compositionmay be molded in the stick form, but usually it is desired to pour itinto a pan for a more convenient use from a consumer standpoint.

The physical properties of the resulting cosmetic compositions can beconveniently adjusted by an appropriate choice of the matrix componentsand by their amount in the composition.

In another embodiment the matrix of the present invention is used as amaterial for industrial purposes, in particular as for dispersioncontrol, for conditioning of liquids and as a lubricant. Because theproperties of the matrix, such as stiffness and temperature of gelationcan be conveniently tuned by adapting the modification degree of themodified polysaccharide, their matrix of the present invention issuitable for a wide range of industrial applications.

The concentration of the polysaccharide components of the matrix in theaqueous solution typically ranges from 1 to 4 wt.-%, particularlypreferred from 1 to 3 wt.-%. In a particularly preferred embodiment theconcentration of the polysaccharide components of the matrix in theaqueous solution ranges from 1 to 2 wt.-%. It is even more preferredthat this concentration is about 2 wt.-%.

The aqueous solution can further contain other compounds, such as saltsor proteins, in particular water-soluble proteins.

PREFERRED EMBODIMENTS OF THE INVENTION

Modified agarose is prepared from agarose by a synthetic protocol knownfrom the prior art. NaOH solution is added to the reaction mixtureduring the reaction in order to maintain the optimal pH of 10.8 of thereaction mixture and neutralize the carboxylic acids being formed. Asoxidant (2,2,6,6-Tetramethyl-piperidin-1-yl)oxyl (TEMPO) is used, whichin turn, is reactivated with sodium hypochloride NaOCl and in thepresence of potassium bromide KBr as a catalyst (Scheme 1). The additionof NaOH further allows following the reaction progress, and gives aquantitative characterization of the amount of carboxylic acid formed asit compensates the formation of the acidic moiety.

Thus, a relationship between the volume of the added NaOH solution andthe ratio of the disaccharide repeat units in which the primary alcoholgroup was oxidized to the carboxylic acid group I (DPI_(Agarose)) isdescribed by the Equation 1.

$\begin{matrix}{{{\frac{m_{Agarose}}{M_{n_{Agarose}}} \times {DPI}_{Agarose}} = n_{NaOH}},} & (1)\end{matrix}$

wherein

-   m_(Agarose) is the mass of the agarose,-   Mn_(Agarose) is the number average molecular weight of the agarose    used as a starting material, and-   n_(NaOH) is the amount of the added NaOH solution.

At the end of the oxidation reaction sodium borohydride NaBH₄ is addedto the reaction mixture. Thus, the aldehyde groups resulting from anincomplete oxidation of the primary alcohol groups are reduced back tothe primary alcohol groups.

In order to have precise and reliable result, the oxidation reaction wasfollowed by several different techniques. As a first qualitativeanalysis the samples of the reaction mixture were lyophilized andanalyzed by ¹³C MAS-NMR. The obtained ¹³C MAS-NMR spectra show theappearance of a peak at 180 ppm, characteristic for the carbonyl carbonatom of carboxylic acid groups, and the vanishing of the peak at 55 ppmwhich is characteristic for the carbon atom of the primary alcohol group(FIG. 4C).

Infrared spectrometry FTIR was used as a quantitative method. The FTIRspectra of the reaction mixture show the increase of the ratio betweenthe peaks at 1650 cm⁻¹: vibration band of the double bond carbon-oxygen(C═O) of the carboxylic acid group and 1360 cm⁻¹: the vibration band ofthe bond carbon-oxygen (C—OH) of the primary alcohol group (FIG. 4B).The relative amounts of the carboxylic acid groups and of the primaryalcohol groups can be calculated by integrating the peaks of interestedand using the Equation (2).

$\begin{matrix}{{{\% \mspace{14mu} {Oxidation}} = {\frac{\int v_{C = O}}{{\int v_{C = O}} + {\int v_{C - {OH}}}} \times 100}},} & (2)\end{matrix}$

wherein

-   ∫ν_(C═O) is the area under the C═O absorption peak,-   ∫ν_(C—OH) is the area under the C—OH absorption peak and-   % Oxidation is the conversion grade of the oxidation reaction.

The formation of carboxylic acid groups along the polysaccharidebackbone was also monitored by measuring the amount of the NaOH solutionconsumed during the oxidation reaction and afterwards by thequantitative analysis using FTIR spectrometry. In order to compare bothanalytical methods, the amount of NaOH solution added to the reactionmixture was plotted against the calculated conversion grade determinedby FTIR. A linear relationship between these values confirms theaccuracy of both analytical methods and verifies the reproducibility ofthe oxidation reaction (FIG. 4D).

It is assumed that the incorporation of ligands at the primary alcoholgroups of the C6 position on the D-galactose of the agarose repeatingunit leads to a lower shear modulus (G′) and a lower temperature ofgelation. In order to investigate in which manner the introduction ofcarboxylic acid groups on the polysaccharide backbone induces the changeof mechanical and gelation properties the authors of the presentinvention conducted a systematic rheological study of the gels.

The gelation point, also named temperature of gelation, is determined bythe equality of the shear storages to the loss moduli at a constantshear frequency and deformation with decreasing the temperature as shownin FIG. 5A. In comparison to the technique of the inverted bottle thetemperature sweep is an objective measure that gives a more precisevalue. As can be readily recognized from FIG. 5C an increase ofmodification degree of a polysaccharide lowers its gelation temperaturein a linear way. In particular, the gelation temperature of nativeagarose is 40° C. (not shown in FIG. 5C), while the gelation temperatureof modified agarose having 93% modification is 5° C.

This phenomenon can be used for tuning of the gelation temperature ofthe gel in order to adapt it to a particular application. Therefore,also the gelation temperature of the matrix of the present invention canbe efficiently tuned by adjusting the modification degree of themodified polysaccharide.

Moreover, the agarose forms a hydrogel that has a hysteretic behavior,i.e. the formation of physical crosslinked points happened at a lowertemperature than the breakdown of the gel. This feature of the gel leadsto an improved thermal stability of the corresponding matrix, and makesit a promising material for biological applications.

The gel stiffness can be characterized by doing a frequency sweep, shearof the gel at a constant deformation and temperature with a frequencyincrease. In rheology a gel state can be defined as state where theshear and loss moduli are both independent on the shear frequency, FIG.5C. The modification of the polysaccharide backbone induces a lowershear and loss moduli which results in a weaker gel. This weakening ofthe polymer network seems to be related to the incorporation ofcarboxylic acid groups, as the loss of primary alcohol groups results ina linear loss of stiffness (FIG. 5D).

The versatile stiffness of these gels can be compared to the humantissue stiffness. Moreover, the values of the shear modulus of thematrix of the present invention are also comparable to those of humantissues. The shear modulus of the cell surrounding has been reported tovary from 10⁵ Pa for bones down to 10¹ Pa for nerves. The order ofmagnitude of the gel stiffness covered by the different proportion ofmodification ranges from 10⁴ Pa for a 2% w/v gel of native agarose to10² Pa for a 93% modified gel of the same concentration. Thus,adaptation of the modification degree of the modified polysaccharideallows an efficient adaptation of the resulting ECM for a wide range ofhuman tissues.

It is known that the agarose backbone folds in an α-helix and that thegel is formed by the aggregation of these helices. The loss of rigidityof the different gels for a given concentration could be attributed to aloss of aggregation of the α-helix. Moreover, the loss of the gelturbidity attributed to the amorphous structures formed by theaggregates reinforces the hypothesis of a loss of crosslinked point(FIG. 5E).

It has been suggested in the literature that the physical crosslinkedpoint formed in the agarose hydrogel can be assimilated as sphericalnanoparticles and then be characterized with static light scattering(SLS). It appears that the increase of the modification degree along thepolymer backbone dramatically decreases the size of this crosslinkedpoint and also their polydispersity resulting in smaller aggregates of asmaller size distribution, see FIG. 6F. These results support thehypothesis of less aggregation of α-helix, but the loss ofpolydispersity is also a quantification of the organization of theaggregate. The decrease of the polydispersity reveals that theaggregates are organized in a more regular shape.

On the other hand, the measurement of the zeta-potential givesinformation of the mobile charges present on the polymer surface. Itappears that the increase of the modification is linearly proportionalto the increase of the zeta potential, see FIG. 6E. The increase of thezeta-potential transcript the incorporation of repulsion charges betweenthe polymer chains that might disrupt the aggregation of the α-helixdomains.

In the past, the α-helix of the agarose has been characterized by usingcircular dichroism (CD) spectroscopy. The native agarose CD spectrum iscomposed of a single peak at 185 nm that is characteristic for anα-helix conformation of the secondary structure. In order to understandthe gelation mechanism of the modified agarose, CD has been measured forseveral modified agarose preparations having different degrees ofmodification. It appears that the peak characteristic for an α-helix isshifted to higher wavelength and a new peak appears at 203 nm, see FIG.6A. The hypothesis of a lower number of α-helix aggregate will have leadto a reduced ellipticity in the α-helix area of the spectrum, but theshift of the spectrum to another area suggest a change of the folding ofthe modified agarose.

The analogy of the conformation of polysaccharides with the folding ofproteins gives an indication of the new secondary structure of themodified agarose. Indeed, the peak in the area of 203 nm corresponds tothe organization of proteins in β-sheet. The consistent study ofdifferent modified gels highlights that the appearance of the new peakis linearly proportional to the amount of carboxylic acid groups alongthe polysaccharide backbone, see FIG. 6C. The modification of theprimary alcohol group to the carboxylic acid group clearly induces achange of the secondary structure into the agarose that is still able toform a gel at low temperature.

The CD spectrum of the modified agarose has been measured at twodifferent temperatures, at 5° C. where the modified agarose is below itssol-gel transition temperature and at 90° C. above its gel-soltransition temperature. It appears that at high temperature the new peakat 203 nm vanishes. The modified agarose forms a gel that is temperaturedependent, whereby the temperature dependency of the new peak suggestthat the gel dependency is now driven by this new organization and notanymore by the aggregation of the α-helix.

Importantly, agarose is not the only polysaccharide which exhibits anα-helix structure. Indeed members of the carrageenan family, forinstance K-carrageenan are also known to organize in α-helices and alsoto form physical gels that are temperature-dependent.

The authors of the present invention carried out the same spectroscopystudy to characterize the k-carrageenan that has been oxidized at thesame position, according the same protocol than the agarose. The peak ofthe unmodified k-carrageenan and unmodified agarose are of opposite signdue to the different helix rotation, but both of them are in the samewavelength. The CD curves obtained for the modified k-carrageenanexhibit the same new peak at the same wavelength of 203 nm, see FIG. 6D.The possibility to obtain the same CD behavior for two polysaccharide ofthe same family implies that the modification of this primary alcoholgroups in theses polymers can be a general method to induce a β-sheetlike structure in polysaccharides.

In order to validate these hypotheses a molecular dynamic (MD)simulation of two polysaccharides backbones over 15 ns was ran. Paststudies on polysaccharide have demonstrated the relevance of the MDsimulation. It is of high importance to validate the model of the neworganization of the modified agarose. The root mean square deviation(RDSD) of the geometry from its initial position shows that after 2 ns astable conformation is obtained. Importantly, this conformation remainsstable until the end of the simulation, see FIG. 7A. In order tocharacterize the distance between the two polysaccharides strands theformation of hydrogen bond (H-bond) between these two strands has beencalculated. The formation of an H-bond is driven by the distance of anelectronegative atom to a hydrogen atom. Each H-bond on each frame hasbeen summed and this sum has been plotted against the time. Therefore astable H-bond sum transcript the non presence of any H-bond, see FIG.7B.

This calculation shows that the native agarose is able to form H-bondall along the simulation, but the totally modified agarose strands donot form any H-bonds after 5 ns, highlighting that the two polymerchains are too far from each other in order to interact together in thismanner. The non interaction of the two polymer strands confirms the dataobtained from the zeta potential that show repulsion forces between thetwo strands.

The main question that has to be answered by the simulation is thephysical and geometrical possibility to form a β-sheet structure. Inpast studies the conformation of the sugar cycles of the polymer chainhas been analyzed using a Ramachandran plot. The dihedral angle of theglycosydic linkage can be plotted and compared with the cartographyobtained for protein folding. The native agarose has its dihedral angleformed by the anhydro-galactopyranose/galactose bound of the chain 1 andgalactose/anhydro-galactopyranose bound of the chain 2 in the α-helixdomain, see FIG. 7C. The same bounds on the modified agarose have theirdihedral angles in the β-sheet area of the plot, see FIG. 7D. The changeof area of the dihedral angles that switch from the α-helix position, tothe β-sheet location show that this suggested organization from theother analysis is possible.

The animations created by the software give an idea about the polymerschains position along the time of the simulation. The conformation ofthe polymer of the first frame has been obtained from the X-ray data ofthe agarose. The two polymers have been modeled with the same initialconformation. It appears that the chains of the native agarose arestaying together along the simulation time, but the chains of themodified agarose are repulsed from each other and position theircarboxylic acid groups opposite to each other.

Thus, the MD simulation attests the possibility of this β-sheetconformation. As for the CD data, the plot of the dihedral angles isbased on the theory developed for proteins. The use of the specific areaof the Ramachadran plot in order to describe the folding of protein hasonly been used so far, for proteins. As the organization of the chainsof polymer changes from an aggregation of α-helix to a β-sheet, theimpact on the macrostructure of the material should be non negligible.

The authors carried out an investigation of the macrostructure of thegel by using environmental scanning electron microscopy (ESEM), see FIG.8, A1 to D1. The highest difficulty in the study of gels is thedifficulty to reveal the hydrated structure of the polymer network.Therefore freeze dried sample have been used for the ESEM. The change ofstructure between the native and the modified agarose shows a radicalchange of their structure. The unorganized fibers of the native agaroseleave the place to a highly ordered fibril structure as the percentageof modification of the polymer backbone is increased. The unorganizedfibers are coupled with small ordered area in the 28% modified sample.The 60% modified gel is more organized and reveals still small domainsof unorganized fibers, but the 93% modified gels is totally organized insheets.

In summary, the authors of the present invention found that themodification of a polysaccharide leads to a change of its tertiarystructure.

This new organization of polysaccharide is surprising. The imagesobtained by the authors validate the hypothesis of a new foldingstructure but moreover confirm the β-sheet organization of the polymerchains. The CD spectroscopy showed that the modification of the primaryalcohol groups of the D-galactose of the agarose repetition unit leadsto the same change of folding of the secondary structure.

The structure of the k-carrageenan hydrogels was imaged by following thesame protocol as for the agarose. The ESEM image reveals that themodification of the k-carrageenan primary alcohol groups leads to thesame highly organized structures forming thin sheets of the same kind asfor 93% modified agarose gels.

In summary, the authors showed for two different polysaccharide of thesame family that the same change of organization takes place whenprimary alcohol groups are oxidized to carboxylic acid groups. Theseresults encourage the possibility of a general method for modifyingpolysaccharide organization and predicting their secondary structurebased on the protein models already existing, see FIG. 8, A4, A5, D4 andD5.

Cells which are cultivated on two-dimensional substrate show adependence on the roughness of the substrate. Therefore the surface ofthe gel has been characterized in a semi-dry state by using atomic forcemicroscopy (AFM). AFM can reveal the roughness of the surface. Theheight pictures highlight a loss of the relief, as the modification isincreased, see FIG. 8, from A2 to D2. The tri-dimensional reconstructionallows visualizing clearly the loss of the roughness, see FIG. 8, fromA2 to D2. The mathematic calculation of the roughness of the gel surfaceappears to be linearly related to the amount of carboxylic acid on thepolymer backbone, see FIG. 8E.

The loss of the roughness can be explained by the smaller crosslinkedpoints, aggregates calculated with the light scattering experiment, thatare formed in the modified gels. The surface is smoother and theorganization in sheets shown by the ESEM correlates with the explanationof a smoother gel that has fibers which are more organized in aunilateral direction.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reaction mechanism for a polysaccharide modification.

FIG. 2 shows examples of polysaccharides.

FIG. 3 shows a scheme illustrating the strategies for preparation ofmodified polysaccharides. In the box on the left hand side modificationsmade by the supplier are shown. In the box on the right hand side twoblendings made by the final user are represented.

FIG. 4A Shows a reaction mechanism of D-Galactose primary alcoholoxidation into a carboxylic acid.

FIG. 4B shows FTIR spectra of the modified agarose (solid line) and thenative agarose (dotted line).

FIG. 4C shows ¹³C-MAS-NMR spectra of the modified agarose (black) andthe native agarose (grey).

FIG. 4D is a plot of the percentage of modification as determined byFTIR against the amount of NaOH solution added to the reaction mixtureas a mean of 12 reactions. Error bars represent the standard deviation.

FIG. 5A shows a temperature sweep of native agarose (dots and triangles)and 60% modified agarose (squares and rhomboids), dots and rhomboidsrepresent G′ and triangles and squares represent G″.

FIG. 5B shows a frequency sweep of native agarose (dots and triangles)and 60% modified agarose (squares and rhomboids), dots and rhomboidsrepresent G′ and triangles and squares represent G″.

FIG. 5C represents a plot showing the relationship between thetemperature of gelation of modified agarose and the percentage of itsmodification. The error bars represent the standard deviation.

FIG. 5D is a diagram showing the shear modulus G′ of agarose sampleshaving different percentage of modification. The error bars representthe standard deviation.

FIG. 5E Photographs of gels containing 2% w/v respectively: A 93%modified agarose; B 60% modified agarose; C 28% modified agarose; Dnative agarose.

FIG. 6A shows a CD spectrum of native agarose (black) and 93% modifiedagarose (grey).

FIG. 6B shows a CD spectrum of 93% modified agarose at 5° C. (dashed)and 93% modified Agarose at 90° C. (solid).

FIG. 6C shows CD of agarose gels having a different degree ofmodification of at 203 nm. Error bars represent the standard deviation.

FIG. 6D shows CD spectra of native agarose (dashed), 93% modifiedagarose (dashed, one point), native k-carrageenan (dashed, two points),93% modified k-carrageenan (dotted).

FIG. 6E shows a plot of zeta potential (mV) of modified agaroses vs.their degrees of modification. Error bars represent the standarddeviation.

FIG. 6F shows plots of polydispersity (black) and size (grey) measuredby light scattering of diluted solutions of different modified agarosesvs. their degrees of modification. Error bars represent the standarddeviation.

FIG. 7A shows a RMSD from the first frame for native agarose (dashed)and 100% modified agarose (solid).

FIG. 7B represents the sum of cumulated H-bonds between the two strandof polysaccharide during the MD simulation for native agarose (black)and 100% modified agarose (grey).

FIG. 7C shows a Ramachandran plot of native agarose AG link in chain 1and GA link in chain 2.

FIG. 7D shows a Ramachandran plot of totally modified agarose AG linkchain 1 and GA link in chain 2.

FIG. 7E shows the MD simulation for native Agarose (left) and from themiddle of the simulation (right).

FIG. 7F shows the MD simulation for totally modified Agarose (left) andfrom the middle of the simulation (right).

FIG. 8. Column A: native polysaccharide, Column B 28% ofmodification-Row 1 is the ESEM of 2% w/v freeze dried agarose gel; Row 2is the AFM picture of the height of agarose gels, Row 3 is the 3Dreconstruction of AFM pictures.

FIG. 8. Column C: 60% of modification, Column D 93% of modification-Row1 is the ESEM of 2% w/v freeze dried agarose gel; Row 2 is the AFMpicture of the height of agarose gels, Row 3 is the 3D reconstruction ofAFM pictures; Row 4 is the ESEM of 2% w/v freeze dried k-carrageenangels at low magnification; Row 5 is the ESEM of 2% w/v freeze driedk-carrageenan at high magnification.

FIG. 8E is a diagram showing the surface roughness of the AFM sample ofagarose gels plotted against the percentage of modification.

EXAMPLES

The following non-limiting examples will illustrate representativeembodiments of the invention in detail.

Methods Description a) Infrared Spectroscopy

FTIR spectra have been recorded on a Brucker Vector 22 FT-IRspectrometer in KBr pellets at 20° C. The pellets were prepared with 2mg of the substance in 200 mg of KBr, then grinded and pressed under apressure of 10 tons press for 10 min.

b) Nuclear Magnetic Resonance

Magic Angle Spinning NMR spectra were recorded at room temperature (20°C.) in the solid state using a Brucker Avance DRX 500 spectrometer. Forthis purpose freeze dried samples were put in a ceramic holder andspined at 7500 U/s.

c) Rheology

Rheology experiments were performed with a MCR rheometer Anton PaarPhysica MCR 301 equipped with a Peltier temperature cell. Sample wereprepare as 2% w/v in deionized water, heated at 90° C. and stirred for10 min until a clear solution was obtained. The liquid was then pouredon the rheometer plate, pre-heated at 80° C., using a pipette. Thesolution was allowed to stabilize for 10 min before the recording wasstarted. A plate tool from Anton Paar: PPR25 was used for allexperiments. Sol-gel transition and frequency sweep were made using thesame sample in a single cycle: 10 min equilibrium at 80° C., coolingdown to 5° C. in 30 min and record of G′ and G″ every 1.5° C. at 1 rad/swith a deformation of 10%, stabilized at 5° C. for 30 min, heated up at37° C. and stabilized for 30 min then the frequency sweep was recordedat 37° C. by increasing the rotation frequency from 0.01 rad/s up to 10rad/s over 30 min with a deformation of 10%. Sol-gel transitiontemperature was calculated as the temperature where tan 8=0.

d) Circular Dichroism

Circular dichroism spectra were obtained using a Jasco J-810spectropolarimeter equipped with a Peltier temperature cell JascoPFD-425S. Solution of 0.15% w/v of agarose was made in Milli-Q water at90° C. for 15 min then solution have been cooled down to 5° C. in the CDchamber for 30 min prior measurement. Each spectrum was recorded threetimes and the obtained spectra were summed together. Each spectrum for agiven modification is a mean of three batches obtained by threedifferent syntheses.

e) Dynamic Light Scattering

Dynamic light scattering (DLS) measurements were carried out on aBeckman Coulter Delsa™ Nano C particle analyzer with a polystyrenecuvette of 1 cm. Agarose was dissolved in Milli-Q water at 90° C. for 15min to obtain a 0.15% w/v solutions that was cooled down at roomtemperature for a day. The light scattering was done at 15° C. andsamples were equilibrated for 30 min prior to measurement. For eachvalue three measurements were done and an average was calculated. Eachpoint is a mean of three batches obtained by three different syntheses.

f) Zeta Potential

Zeta potential was measured on a Beckman Coulter Delsa™ Nano C particleanalyzer. The same solutions were used as for the light scatteringexperiment. Measurements were made in a flow cell that was aligned withthe laser prior every measurement. Each measurement was made three timesand an average was calculated. Each spectrum for a given modification isa mean of three batches obtained by three different syntheses.

g) ESEM

ESEM images were obtained with a ref agarose gels. 2% w/v solutions wereprepared and 2 ml of these solutions were freeze-dried for 24 hoursunder 0.1 mbar vacuum in a 5 ml glass vial. The samples were verticallycut and the inside of the sample was imaged at different magnification.Images shown here are representative images of different areas of agiven sample at different magnification, which were reproduced withthree different gels prepared from different batches.

h) AFM

AFM images were obtained with a scanning probe microscope VeecoDimension 2100. The samples were prepared on a 3 mm microscopic glassholder that was previously passivated. The glass slide was washed with0.1 M NaOH solution and dried in the oven. The dry slides were thenpassivated with a few drops of dichloromethylsilane. Two slides weresandwiched together to have a uniform passivation. After 10 min theslides were washed with water and the excess of dichloromethylsilane waswashed away with soap after what the slides were dried. Slides side wasprepared in a hydrophobic way. Agarose samples were prepared as 2% w/vgels and 25 μl of the obtained solution was poured on an unmodifiedglass slide. A dichloromethylsilane passivated slide was then adjustedon top of the solution. Slides of 0.5 mm were put as spacers between thehydrophobic and the normal glass slide, the whole montage was thenallowed to gel for 30 min at 4° C. The upper slide (hydrophobic) wasafter that removed and a thin layer of agarose gel was obtained. Thisgel was then allowed to stabilize at room temperature for 30 min beforemeasurement in order to avoid any shrinkage or dilatation of the gelduring the measurement.

i) Molecular Dynamic Simulations

MD simulations were done using the Desmond package of the Maestro,Version 8.5 from Schrödinger. Initial conformation was been obtainedfrom the X-ray structure of the agarose that was downloaded from theprotein database (PDB) library. Modified agarose was drawn from the PDBfile directly inside the Maestro software. Implicit water model wasbuild using the Desmond tool, resulting in a 10 Å square box build byfollowing the TIP3 solution model. The simulations were run in the modelNPV at 300° K at atmospheric pressure for 15 ns. Analysis of the resultswas done using the VMD software and the tools available in the standardpackage.

j) Elementary Analysis

Samples have been analyzed on an element analyzer Elementar Vario EL

Example 1. Modification of Agarose

Agarose type I was obtained from Calbiochem.(2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO), NaOCl, NaBH₄, NaBr,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and2-(N-morpholino)ethanesulfonic acid (MES) buffer were obtained fromSigma Aldrich and used as received. Solution of 0.5 M NaOH as well assolution of 5 M HCl were freshly prepared every three-month. Ethanoltechnical grade was used without any further purification. Deionizedwater was used for non-sterile synthesis.

Agarose was modified under sterile conditions: all the chemicals weredissolved in autoclaved water and filtered with a 0.2 μm filter. All theglassware was autoclaved and the reaction was conducted under a laminarflow. Agarose (1 g) was autoclaved in MilliQ water. Autoclaved agarosewas poured into a 3 necked round bottom flask, which was used as areactor. A mechanical stirrer was adapted to one of the neck. A pH-meterwas adapted another neck of the round bottom flask. The reaction mixturewas then cooled down to 0-5° C. and vigorously stirred. TEMPO (0.160mmol, 20.6 mg) was added, NaBr (0.9 mmol, 0.1 g) and NaOCl (2.5 ml, 15%solution) was as well poured inside the reactor. The resulting solutionwas adjusted to pH=10.8 with HCl and NaOH solution. The pH wasmaintained at 10.8 by adding NaOH solution. At the end of the reactionNaBH₄ (0.1 g) was added and pH=8 was reached. The solution was stirredfor 1 hour and NaCl (0.2 mol, 12 g) and ethanol (500 ml) was added. Theagarose was precipitated and extracted in a funnel. The two layers werethen filtered on a frit glass. The agarose was then dialyzed in SpectraPore 4 membranes, MWCO=12−14000 for 2 days and the water was changed twotimes. Prior dialysis, the membranes were left overnight in a 70%ethanol solution, 2 hours before use they were rinsed in autoclavedwater. Finally, the product was put on a freeze-drier Christ LD 2-8 LDplus at 01 mbar for the main drying and at 0.001 mbar during thedesorption phase. Samples were put in round bottle flask and freezed inliquid nitrogen bath on a Rotary evaporator modified for this purpose.Thin layer of frozen solution was obtained on the flask wall reducingthe lyophilization time.

Example 2. Modification of k-Carrageenan

K-carrageenan was obtained from Sigma Aldrich and used as such.K-carrageenan was objected to a TEMPO-mediated oxidation, according tothe synthetic protocol of Example 1.

The resulting modified k-carrageenan was dialyzed and freeze-dried, asspecified in Example 1 above.

Example 3. Blending of Modified Agarose with Unmodified Agarose

Blends of modified agarose and native agarose in different proportionswere prepared. The resulting blends were studied using CD spectrometry.The obtained results indicate that the blending of two polysaccharideslead to the same change in tertiary structure as the modifiedpolysaccharide. This suggests that these two polysaccharides aremiscible and can be used for engineering new matrices.

The rheology studies show a specific behavior of the gel. Indeed theblended gels have a higher shear modulus than the unmodified gel ofagarose. These results suggest that the organization of the modifiedchains with the unmodified chains follows a new mechanism, that isdifferent from modified agarose.

The ESEM image illustrates the structure of the gels and reveals adifferent organization of the fibers than for the unmodified agarose. Aswell for the surface roughness, the roughness of the blended gels is notdependent of the proportion suggesting a new mechanism of organization.

Example 4. Covalent Biding of Peptide to Modified Agarose

Peptide GGGGRGDSP (SEQ ID NO: 3) was obtained from PeptideInternational.

Functionalization of agarose with the G₄RGDSP peptide (SEQ ID NO: 3) wasdone by EDC peptide coupling. Agarose (30 mg, 0.25 μmol) was dissolvedin autoclaved water; all the chemical and buffer were sterilized on a0.2 μm filter, MES buffer was added and the solution reached a pH=4. Thepeptide (500 μg) was added thereto, followed by EDC (200 mg) and theresulting solution was stirred for two hours at 40° C. to avoid any gelformation, The solution was then dialyzed in Spectra Pore 4 MWCO=12-14kDa, whereby water was changed three times. Subsequently, the sample wasfreeze-dried. Prior dialysis the membranes were left overnight in a 70%ethanol solution, 2 hours before use they were rinsed in autoclavedwater.

Example 5: Use of Modified Extracellular Matrix for Biological TestsCell Culture:

Modified gels were prepared at a 2% w/v concentration in DMEM media andheated at 60° C. for 30 minutes in order to avoid any degradation of theRGD peptide. The temperature was adjusted to 37° C. and the culturemedia was completed with usual nutrients and cytokins. Humanchondrocytes were obtained from ATCC and used between passage 3 and 5.Solution of agarose was mixed with the cells and then seeded in a 48wells plate. The plate was then stored at 4° C. for up to 30 min inorder to allow the sol-gel transition to occur. The plates were thencultivated in a incubator at 37° C., 4% CO₂ for two weeks.

Cell Shape Factor.

All images were taken on an Axio Observer A1, from Carl Zeiss, equippedwith a differential interference contrast (DIC) filter. The images weretaken after 1, 5 and 14 days at different magnification in differentarea of the sample. The cell perimeter and cell area were then measuredand the cell shape factor (CSF) was calculated. The results are a meanof three different batch of peptide modified agarose which wasreproduced two times, which represent a total of 6 wells. Per well morethan 100 cells were measured in order to have a meaningful CSF.

Real Time PCR:

At 7 days, 14 days and 21 days of cell culture in the gel, the media wasremoved and the gels were frozen at −80° C. overnight. The solidsobtained were then crushed and digested with a CT AB buffer. Thenextract using the Qiagen kits.

1-19. (canceled)
 20. A plastic surgery implant consisting essentially ofa matrix comprising: a modified polysaccharide consisting of repeatingdisaccharide units, whereby in at least 11% of the disaccharide unitsone primary alcohol group is oxidized to a carboxylic acid group,wherein the matrix further comprises an unmodified polysaccharidecomponent to adjust the mechanical properties of the matrix, and,wherein the shear modulus G′ of the matrix is in the range of from 10 Pato 10⁷ Pa.
 21. The plastic surgery implant of claim 20, wherein themodified polysaccharide is selected form the group consisting of amember of the carrageenean family, hyaluronic acid, heparin sulfate,dermatan sulfate, chondroitin sulfate, alginate, chitosan, pullulan, andagarose.
 22. The plastic surgery implant of claim 20, wherein themodified polysaccharide is agarose.
 23. The plastic surgery implant ofclaim 20, wherein the unmodified polysaccharide is selected from thegroup consisting of a member of the carrageenean family, hyaluronicacid, heparin sulfate, dermatan sulfate, chondroitin sulfate, alginate,chitosan, pullulan, and agarose.
 24. The plastic surgery implant ofclaim 20, wherein the unmodified polysaccharide is agarose
 25. Theplastic surgery implant of claim 20, wherein the shear modulus G′ of thematrix is in the range of from 10 Pa to 100 kPa.
 26. The plastic surgeryimplant of claim 20, at least 20-99% of the disaccharide units oneprimary alcohol group is oxidized to a carboxylic acid group
 27. Theplastic surgery implant of claim 20, wherein a weight ratio of themodified polysaccharide to unmodified polysaccharide in the matrix isgreater than 10 wt % of a modified polysaccharide.
 28. A method forreconstructive and cosmetic surgery in diverse body regions, the methodcomprising implanting in plastic surgery a plastic surgery implantaccording to claim 1.